The ability to characterize entire classes of proteins based on activity would greatly accelerate both the assignment of protein function in the genome era and the identification of new biomarl<ers and therapeutic targets for the diagnosis and treatment of human disease. With this goal in mind, we have created innovative chemoproteomic platforms to profile protein activities en masse in native biological systems. In the past grant period, we advanced and applied these platforms and the following major achievements were attained: 1) discovery and inhibition of deregulated, pro-tumorigenic lipid hydrolases; 2) development of an integrated proteomic platform to discover novel forms of caspase-kinase crosstalk that regulate cancer apoptosis; 3) discovery of a novel post-translational modification that couples glucose uptake to glycolytic remodeling and the production of pro-tumorigenic metabolites; 4) development of a tag-free activity-based protein profiling (ABPP) platform to quantify and functionally characterize reactive cysteines in cancer; and 5) advancement of tag-free ABPP to optimize the activity and selectivity covalent kinase inhibitors as anti-cancer drugs. In this renewal application, we will build upon our past achievements to test the following major hypotheses of high significance to cancer and chemical biology: 1) lipid hydrolases form key bridges between metabolic and signaling pathways that promote tumorigenesis; 2) crosstalk between proteolytic and phosphorylation pathways regulate cancer cell survival and create tumor-specific biomarkers for monitoring cancer therapy and resistance; 3) 3-phosphoglyceryl-lysine (pgK) modifications constitute an intrinsic feedback pathway to remodel glycolytic output and stimulate tumor growth; 4) electrophile/oxidation-sensitive cysteines regulate cancer cell survival in response to oxidative stress; and 5) covalent kinase inhibitors optimized by tag-free ABPP will show enhanced on-target activity and reduced off-target interactions, thereby expanding their therapeutic window as anti-cancer drugs. The ultimate goal ofthis application is to Identify key biochemical pathways that support human cancer growth and malignancy. The molecular components of these pathways may represent new biomarkers and drug targets for cancer.